Microscopes are well-established instruments for observing minute biological structures and processes. This field of art includes fluorescent microscopy, which allows a user to achieve a high degree of target specificity and image contrast for biological specimens. Conventional fluorescent microscopes employ the basic illumination scheme shown in FIG. 1. In FIG. 1, a viewing device 10 (such as a Charged Coupled Device (CCD) or camera or eye) is used to observe a biological sample 22 which rests upon stage 20. The viewing device 10 includes a viewing path through an image forming tube lens 26, an emission filter 34, a dichroic mirror 30, and an objective lens 24.
A lamp 40 emits an excitation light 50 that has passed through the collector 32 and which then passes through the conjugate image plane located at the field diaphragm 42. The excitation light 50 continues through the projection tube lens 28 and an excitation filter 36. The excitation light 50 is of a wavelength that is reflected downward by the dichroic mirror 30, after which it passes through objective 24 to thereby reach sample 22 where the excitation light 50 stimulates a fluorescent marker in the sample 22. The sample 22, thus excited, emits a fluorescent emission which is of a wavelength permitted to pass through the dichroic mirror 30, and which is viewed by the viewing device 10.
Known methods for illuminating a biological specimen in fluorescent microscopy utilize an even excitation illumination across the entire field of view. For instance, Kohler illumination applies an even excitation light across an entire sample. Problems arise from these known systems of illumination, however, as there are situations where it may be better to vary the intensity of illumination over different regions of the specimen.
For instance, during live cell imaging only a portion of the field of view is of interest and unnecessary exposure of light to living tissue in the remainder of the field can result in photo-toxicity and/or photo-bleaching. An additional problem occurs when a bright object is located in front or in back of a structure of interest, making it impossible to clearly view the object of interest.
Photo-toxicity is a particularly troubling problem in live cell fluorescence microscopy. First, fluorescence microscopy requires very intense excitation illumination that is typically many orders of magnitude brighter than that used for conventional brightfield microscopy. Photo-toxicity due to an intense excitation illumination light is often the ultimate limitation in live cell fluorescence imaging. Secondly, all fluorescent dyes produce toxic free radicals upon illumination with excitation light.
The current invention minimizes photo-toxicity and photo-bleaching through control of the excitation illumination, for instance, by modulating the intensity of the excitation light through at least one of spatial modulation and temporal modulation. In spatial modulation, different regions of the specimen are simultaneously illuminated with different intensities of excitation light based on the needs and interests of the observer. In temporal modulation, different regions of the specimen are scanned by an excitation light of varying intensity over a time period.
Electronic image sensors and detectors have replaced photographic cameras as the primary means to record images in wide-field fluorescence microscopy. An inherent weakness of current digital detectors is a limited ability to record images with high variations in brightness. This becomes a serious problem when sub-regions of a sample are much brighter than the regions of interest. The bright regions saturate the detector while the regions of interest are inadequately illuminated. These problems are discussed below in relation to FIGS. 2A-2C.
Image quality depends upon the proper biasing of illumination brightness so as to stay within the optimal detection range of the CCD detector. Under-excitation results in a poor signal-to-noise ratio. An example of under-excitation is shown in FIG. 2A, where the image of the sample was acquired under insufficient illumination. Other problems result from over-excitation. As shown in FIG. 2C, the sample being viewed was over-excited, resulting in pixel saturation at the CCD. Note that the image is distorted, leaving no plausible manner in which to perform quantitative analysis.
As shown in FIG. 2B and so long as the sample being viewed has relatively low variations in brightness between various portions of the sample, it should be possible to find a proper level of excitation illumination that can produce a qualitatively pleasing and quantitatively accurate image. However, with current microscopes, and when a sample's emitted brightness possesses moderate-to-high variability, it has been heretofore impossible to select a single proper level of excitation illumination (i.e., exposure duration) that will produce optimized imaging for each aspect of a sample being viewed.